22.214.171.124. in case of lipid-rich samples (e.g. whole larva)
An additional purification step is usually required as lipids may interfere in the ligand-binding and the ammonium sulphate precipitation of the antigen-antibody complex. A rapid and efficient purification is the separation of free ecdysteroids from polar or apolar compounds by means of reverse-phase liquid chromatography on disposable reverse-phase columns (SepPak C18, Waters, Waters, WAT051910).
1. Evaporate methanol from sample (after centrifugation to remove impurities) by vacuum centrifugation.
2. Resuspend sample in 30 % methanol.
3. Activate SepPak C18 cartridge mounted on a 1 ml syringe by slowly passing pure methanol.
4. Equilibrate column by passing 1 ml 30 % methanol.
5. Load sample on column
6. Pass 2 x 1 ml 30 % methanol.
This eluate contains polar ecdysteroid conjugates that can be discarded, or if of interest, analysed after enzymatic digestion (Helix pomatia juice).
7. Pass 2 x 1 ml 60 % methanol through column and collect eluate in a 5 ml disposable glass test tube.
This eluate contains the free ecdysteroids
8. Discard column.
9. Evaporate solvent from the free ecdysteroid fraction samples by vacuum centrifugation.
10. Re-dissolve in 100 % methanol for the RIA procedure.